【作者】 王燕；

【导师】 王柯敏； 何晓晓；

【作者基本信息】 湖南大学 ， 分析化学， 2006， 硕士

【摘要】 随着纳米技术的不断发展和渗透,纳米颗粒已经在材料科学、生物化学、医学等领域得到重大应用。其中基于纳米颗粒来研制化学生物传感器已经成为纳米技术研究中的一个新的热点,这主要是由于纳米颗粒传感器具有体积小、分辨率高、响应时间短、所需样品量少等优点。本论文拟在本研究小组所构建的硅壳荧光纳米颗粒技术平台的基础上,进一步开展基于硅壳荧光纳米颗粒的纳米传感器的制备及其在生物医学上的应用研究,本论文主要包括以下三方面工作:1、内参比pH纳米传感器的制备并用于抗肿瘤药物作用的Hela细胞内pH变化的测量利用反相微乳液技术发展了一种同时包埋pH敏感染料异硫氰酸荧光素(FITC)和参比染料联钌吡啶(RuBpy)的基于硅壳荧光纳米颗粒的内参比pH纳米传感器。该内参比pH纳米传感器具有大小均匀、尺寸在50 nm左右、在水溶液中分散性好、抗光漂白能力强、染料泄漏少等优点,对pH线性响应范围为4.4~7.4。利用该内参比pH纳米传感器尺寸小可以通过细胞的内吞而无损伤地进入细胞内的优势,进一步结合流式细胞学分析方法,对不同抗肿瘤药物作用的Hela细胞内的pH变化进行了分析,结果表明:不同抗肿瘤药物作用后的Hela细胞内pH的变化情况并不一样,有的作用后引起了细胞酸化,有的作用后引起了细胞碱化,有的作用后基本上不引起变化。同时,采用激光共聚焦显微镜对吞噬了内参比pH纳米传感器的抗肿瘤药物作用的单个Hela活细胞进行了成像,可以明显观察到内参比pH纳米传感器在不同抗肿瘤药物作用后的Hela细胞内的荧光成像。因此,基于硅壳纳米颗粒发展的内参比pH纳米传感器可以用于细胞内变化pH的实时监测。2、基于内参比pH纳米传感器的Hela细胞内酸化与细胞凋亡的相互关系研究采用上一章发展的pH敏感染料异硫氰酸荧光素(FITC)和参比染料联钌吡啶(RuBpy)同时包埋的具有内参比功能的pH纳米传感器研究了抗癌药物硫酸长春新碱诱导的Hela细胞内酸化与细胞凋亡之间的相互关系。通过利用该纳米传感器对硫酸长春新碱诱导后的Hela细胞内pH变化的活体、原位、实时监测,发现在硫酸长春新碱诱导引起凋亡的Hela细胞中,细胞内的pH值由诱导前的7.11酸化为6.51,并且在一定的浓度和时间范围内,硫酸长春新碱诱导后细胞内的酸化率与诱导药物的浓度和诱导时间成正相关关系。通过进一步比较硫酸长春新碱诱导后的细胞酸化率和细胞凋亡率,证实了经硫酸长春新碱诱导后,Hela细胞内酸化是Hela细胞凋亡的先发事件。这些研究结果的获得有望为利用硫酸更多还原

【Abstract】 With the development of nanotechnology, nanoparticles have been significantly applied in the fields of material science, biochemistry and medicine. The chemical and biological nanosensors based on nanoparticles have become a new focus in the research of nanotechnology, because their advantages such as small volume, high resolution, short response time, few sample and so on. Aimed at the above advanced direction, this thesis was focused on the preparation of nanosensors and application of these nanosensors in the biomedicine, which were on the basis of silica shell fluorescent nanoparticles technology developed by our group. This thesis mainly includes following three works.1.Preparation of ratiometric pH nanosensors and its appication in intracellular pH detection of the anti-tumor drugs treated Hela cellsThe ratiometric pH nanosensors that contained a pH sensitive indicator fluorescein isothiocyanate (FITC) and a reference dye tris(2, 2′-bipyidyl) dichlororuthenium(II) hexahydrate (RuBpy) had been prepared with O/W microemulsion method. The ratiometric pH nanosensors had an average diameter of 50 nm, good photastability and could be well dispersed in aqueous solution. The linear response range of the ratiometric pH nanosensors to pH was between 4.4 and 7.4. The small size of the ratiometric pH nanosensors allowed it to be easily taken up by cells for noninvasive intracellular pH measurement, so the intracellular pH of different anti-tumor drugs treated Hela cells had been detected by combination with the advantages of the small size of the ratiometric pH nanosensors and the flow cytometry analysis method. The results demonstrated that the intracellular pH change was different when the Hela cells was treated with different anti-tumor drugs, some would caused intracellular acidification, some would caused ntracellular basification, and some would barely caused pH change. The single living Hela cells uptaken with the ratiometric pH nanosensor was also imaged with laser confocal microscope, which were treated with anti-tumor drugs. The results indicated that ratiometric pH nanosensors based on the silica shell fluorescent nanoparticles could be used to real- time monitor the intracellular pH change.2. Research of the relationship of intracellular acidification and apoptosis in Hela cells based on pH nanosensors更多还原